Cytochrome c is an essential protein component of the respiratory apparatus of all mammalian cells. Although it functions in the mitochondia, it is encoded by a nuclear gene. We previously isolated and determined the sequence of several cytochrome c genes from a rat genomic library and made the surprising observation that these genes belong to a multigene family comprising 25 to 30 closely related sequences in the genomes of rat and many other mammals. In addition we discovered multiple forms of cytochrome c mRNA in various rat tissues and determined that these mRNAs are most highly homologous to a 7-member subgroup of genes from the cytochrome c multigene family. We determined the nucleotide sequence of 4 genes from this subgroup. The specific objectives of this proposed research are the following: 1. To isolate and determine the nucleotide sequences of the remaining 3 genes from the 7-member subgroup most closely homologous to cytochrome c mRNAs. This will result in a knowledge of the detailed structures of these potentially active genes. 2. To use S1 nuclease mapping to establish which of these genes are colinear with any of the multiple cytochrome c mRNAs. This will identify the genes most likely to direct the synthesis of mRNAs as well as to determine the structural basis of mRNA heterogeneity. 3. To use transcriptionally active isolated nuclei from liver and various other tissues of rat to map cytochrome c transcription units. These experiments will identify genes synthesizing primary transcripts and determine transcription initiation and termination sites. 4. To use the nuclear transcription system to study the induction of cytochrome c mRNA in response to thyroid hormone. Thyroid hormones increase the rate of biosynthesis of cytochrome c approximately 6-fold in rat liver. These studies will allow us to examine the effects of thyroid hormone on the primary transcription of a specific gene.